Vaccines

ABSTRACT

The invention provides  Streptococcus  vaccine strains (e.g.  S. equi  which is causative of ‘strangles’) comprising the following modifications in their genomes: (i) attenuation of one or more essential biosynthetic genes, (ii) attenuation of one or more genes which encode a haemolytic toxin, or protein involved in the production thereof, plus preferably any one, two, or most preferably three of the following modifications: (iii) attenuation of one or more genes which encode a protein responsible for immune evasion (iv) modification of one or more genes such as to permit serological discrimination of the vaccine strain based on analysis of a protein encoded by said genes, and (v) attenuation of one or more genes which encode an enzyme responsible for recombination repair of the genome. The invention provides inter alia live attenuated vaccine strains which generate an immune response to multiple protective epitopes presented in a context resembling the live pathogen.

TECHNICAL FIELD

The present invention relates generally to methods and materialsconcerning diseases caused by Streptococcal pathogens, and in particularrelating to attenuated strains of Streptococci and use of the same asvaccines.

MICROORGANISM DEPOSIT

A micro-organism deposit in accordance with the Budapest treaty has beenat the National Collection of Type Cultures (NCTC), Health ProtectionAgency, Centre for Infections 61, Colindale Avenue, LONDON NW9 5EQ,United Kingdom under accession number: 13412 on date 26 Sep. 2007 onbehalf of the present applicant by the present inventors.

The depositors have authorised the present applicant to refer to thedeposited biological material in the application.

BACKGROUND ART

Streptococcus is a genus of spherical shaped Gram-positive bacteria.Clinically, individual species of Streptococcus are classified primarilybased on their Lancefield serotyping—according to specific carbohydratesin the bacterial cell wall. These are named Lancefield groups A to T.However the pathogens in these different groups share many similaritiesat the genetic level. For example Streptococcus equi (which is in groupC, and which is the causative agent of equine strangles) shares 80%genome identity with the human pathogen S. pyogenes (which is in groupA, and which is the causative agent of many human conditions includingstrep throat, acute rheumatic fever, scarlet fever, acuteglomerulonephritis and necrotizing fasciitis). Additionally the twoorganisms share many near identical toxins and virulence factors.

Streptococci are further characterised via their haemolytic properties.Alpha haemolysis is caused by a reduction of iron in haemoglobin givingit a greenish color on blood agar. Beta only haemolysis is completerupture of red blood cells giving distinct, wide, clear areas aroundbacterial colonies on blood agar. Other streptococci are labeled asgamma haemolytic.

Strangles is a disease characterised by nasal discharge and fever,followed by abscessation of local lymph nodes. The swelling of the lymphnodes in the head and neck may, in severe cases, restrict the airway andit is this clinical feature that gave the disease ‘strangles’ its name.Morbidity rates of up to 100% are reported and mortality as a result ofdisseminated abscessation ('bastard strangles') may occur in 10% ofcases (Timoney, 1993a). Strangles is one of the most frequentlydiagnosed equine diseases worldwide. Recent outbreaks in Thoroughbredshave further highlighted the need for the development of improvedtherapies. Antibiotic treatment is usually ineffective despite S. equi'ssusceptibility to most antibiotics in vitro. Clinical signs followingtreatment have been reported to abate only until treatment is withdrawn.This relapse is probably due to the lack of sufficient vascularity inthe abscess to enable antibiotic penetration to therapeutic levels andillustrates the importance of the development of an effectivepreventative vaccine (Harrington et al., 2002). Approximately 10% ofhorses that recover from strangles become carriers of the disease,harbouring the infectious agent in chondroids located in the gutturalpouch. These carriers are capable of infecting other naïve horses andcontinue the spread of disease (Chanter et al., 2000, Newton et al.,1997, Newton et al., 2000). Therefore, a major goal of vaccine design isnot only to protect against strangles, but also to prevent developmentof the carrier state.

Progress in the development of an effective strangles vaccine has beenslow. Vaccines against the disease have been known for a long time(Bazeley, 1940 and Bazeley, 1942), but have either proved ineffective orsuffer from undesirable side effects.

Four kinds of vaccines are available: a) vaccines based on classicalbacterins, b) sub-unit vaccines based on the M-protein, an immunogenicprotein, c) Chemically attenuated live Streptococcus equi and d)Genetically attenuated live Streptococcus equi.

Conventional vaccines containing inactivated whole bacteria or extractshave shown little efficacy and often induce adverse reactions (Jorm,1990, Timoney and Eggers, 1985). Classical vaccines based on bacterinsor subunits are e.g. available through Fort Dodge Laboratories andCoopers Animal Health.

Those vaccines that specifically target the M-protein of S. equi, showedpromise in mouse vaccination challenge studies (Meehan et al., 1998),but failed to demonstrate significant protection in horses despite thegeneration of M-protein reactive antibodies (Timoney et al., 1997,Sheoran et al., 2002).

Similarly, a recombinant S. equi hyaluronate associated protein (HAP)vaccine, was partially protective in mice (Chanter et al., 1999), butfailed to prevent the development of strangles in vaccinated horses (N.Chanter unpublished results).

The basis and duration of protective immunity following naturalinfection is not fully understood, but in the majority of animals thatrecover from strangles immunity is believed to last for >5 years (Hamlenet al., 1994; Sweeney et al., 2005; Todd 1910; Woolcock, 1975).

A non-specifically attenuated vaccine strain for intranasal inoculation,the ‘Pinnacle I.N.’strangles vaccine, is marketed by Fort Dodge(Timoney, 1993b, http://www.wyethah.ca/wyeth_equine/pinnacle.html). Thisacapsular strain was derived following chemical mutagenesis to inducerandom mutations throughout the bacterial genome (Timoney, 1993b). Suchnon-defined point mutations are prone to back mutation and thus toreversion to full virulence and although this vaccine may protect up to100% of horses (Timoney, 1993b, Walker and Timoney 2002), it has notbeen licensed for sale in Europe due to safety concerns. These includenasal discharge, lymphadenectasis and a 5% risk of submandibularabscesses following IN vaccination (Timoney, 1993b,http://www.wyethah.ca/wyeth_equine/pinnacle.html). US2006110411 (WYETHFORT DODGE LAB (US)) relates to compositions comprising live, attenuatedS. equi.

Recently, an Intervet live attenuated vaccine strain TW 928 has beenapproved for sale in Europe, marketed as ‘Equilis StrepE’. This strainwas attenuated by the partial deletion of the aroA gene and was 10⁴-foldattenuated during intraperitoneal mouse challenge studies (Hartford etal., 1999, http://www.biosafety.be/EMEA/Table_EquilisStrepT.htm). The TW928 vaccine strain was attenuated in six horses with no signs of diseaseapparent at post mortem examination four weeks after intranasalchallenge (Hartford et al., 1999). Intramuscular vaccination of horseswith strain TW 928 conferred 100% protection from subsequent S. equichallenge. However, severe injection site reactions precluded the use ofthis route for future studies. Further, contamination of needles to beused for the administration of other products with the Equilis StrepEvaccine have also led to abscess formation at the intramuscularinjection site (Kemp-Symonds et al., 2007).

In order to minimise injection site reactions and retain some protectiveefficacy, sub-mucosal vaccination with 10⁹ cfu of the TW 928 strain intothe inside of the upper lip was evaluated. Using this method, smallpustules formed over a period of one week from which the TW 928 straincould be isolated. Horses were 50% protected from intranasal S. equichallenge and a further 25% of vaccinates had reduced clinical signs ofdisease. The presence of these pustules may be critical for thegeneration of an efficacious immune response since on dose reductionreduced injection site reactions correlated with decreased protection(Jacobs et al., 2000).

We have also observed cases of sub-mandibular lymph node abscessation inhorses recently vaccinated with Equilis StrepE. In three of these caseswe have confirmed by genetic analysis that the causal agent was thevaccine strain (Kemp-Symonds et al., 2007; Waller et al., unpublisheddata).

In addition, an undetermined proportion of the 25% of vaccinated horses,which on exposure to virulent S. equi suffer reduced clinical signs maygo on to become carriers of virulent field strains of S. equi withoutbeing diagnosed. Such a scenario is of major concern to diseaseprevention strategies.

Finally, the vaccine suffers from only a 3-month duration of immunity,although boosting of horses vaccinated up to six months previously inthe face of an outbreak has been shown to improve clinical outcome andextends the usefulness of this vaccine.

Overall, ‘Equilis StrepE’ is a promising advance over the Pinnaclestrain (most notably in its lower risk of reversion). However, it isonly recommended for use in horses of high or moderate risk of strangleswhere acquisition of a short duration of immunity is advantageous(http://www.wyethah.ca/wyeth_equine/pinnacle.html). Additionally itsuffers a number of drawbacks as discussed above.

It will be appreciated that novel vaccine strains which could overcomeone or preferably more than one of these drawbacks would provide acontribution to the art.

DISCLOSURE OF THE INVENTION

The present inventors have considered the drawbacks in the prior artabove and have determined that greatest protection against Streptococcalpathogens may be achieved by use of preferably live attenuated strainswhich generate an immune response to multiple protective epitopespresented in a context most resembling the live pathogen.

Such strains are provided herein.

In respect of strangles vaccines, the attenuated vaccines describedherein may offer improved efficacy, intramuscular safety, have agenerally improved safety profile, and may not cause the occasionallymph node abscessation found with the prior art.

In one embodiment there is disclosed herein

A Streptococcus vaccine strain comprising the following modifications inits genome:

-   -   (i) attenuation of one or more essential biosynthetic genes,    -   (ii) attenuation of one or more genes which encode a haemolytic        toxin, or a protein involved in the production thereof, plus        preferably any one, two, or most preferably three of the        following modifications:    -   (iii) attenuation of one or more genes which encode a protein        responsible for immune evasion,    -   (iv) modification of one or more genes such as to permit        serological discrimination of the vaccine strain based on        analysis of a protein encoded by said genes, and    -   (v) attenuation of one or more genes which encode an enzyme        responsible for recombination repair of the genome.

By “attenuation” is meant modification of the sequence of relevant geneand hence impairment of the function or activity of the encoded protein.Preferred attenuations are deletion of all or part of the gene, or theintroduction of substitutions therein. Preferably attenuation isachieved by at least one well-defined irreversible deletion ofsubstantial size.

Those skilled in the art will appreciate that the strains of the presentinvention may combine one or more further attenuations in addition tothose described above.

Some particular aspects and embodiments of the invention will now bediscussed in more detail.

Biosynthetic Genes

The inventors have determined that attenuation of S. equi TW 928 by thedeletion of only one gene may not remove the possibility of strainreversion. Acquisition of homologous DNA from the commensal S.zooepidemicus followed by recombination and repair of the TW 928 genomeremains a small, but unknown risk factor in the use of this strain inequids.

Preferably therefore two essential biosynthetic genes are attenuated.Preferably the genes are partially or fully deleted.

Preferred target biosynthetic genes are those in the aromatic orpyramidine (or purine) pathways. Preferred genes are aroB and pyrC.

Deletion of genes in the aromatic amino acid biosynthetic pathway isknown to attenuate pathogens and has been used to generate the TW 928 S.equi strain and several non-streptococcal vaccine strains(http://www.biosafety.be/EMEA/Table_EquilisStrepT.htm, Ingham et al.,2002, Simmons et al., 1997, Chamberlain et al., 1993, Alexander et al.,1993, Vaughan et al., 1993, Karnell et al., 1992, Newland et al., 1992,Stocker 1990, Izhar et al., 1990).

Purine and pyrimidine biosynthesis have been targets for attenuation ofpathogenic E. coli (Kwaga et al., 1994) and inactivation of pyrC isknown to prevent growth of B. subtilis in minimal media (Waller et al.,2001).

Haemolytic Toxin

As noted above the relevant gene may encode the toxin or a proteininvolved (or more preferably required) for the efficient productionthereof, such that the attenuation reduces the level of toxin produced.Preferably one such gene is partially or fully deleted.

A preferred gene is the sagA gene, essential to production of thestreptolysin S haemolytic toxin. Work in Streptococcus pyogenes hasidentified that injection site lesions could be reduced by inactivationof the SLS haemolytic toxin in this Group A Streptococci (Betschel etal., 1998).

Immunogenicity

The attenuation of genes leading to immune evasion will lead toincreased immunogenicity.

Immune evasion in this context is used broadly to cover situations bothin which an immune response is directly suppressed, and also where it ismisdirected—for example by so-called “superantigens”, which exhibithighly potent lymphocyte-transforming (mitogenic) activity directedtowards T lymphocytes.

Example genes may encode enzymes or other proteins involved inproduction of the hyaluronate capsule.

Preferably one such gene is partially or fully deleted.

A preferred gene is the hasA gene which has been described in relationto Streptococcus pyogenes (Dougherty and van de Rijn 1994, Wessels etal., 1994).

Other preferred genes may encode superantigen toxins. Although these aretoxins in their own right, it is believed that their primary effect maybe to misdirect the immune response. Examples of such genes are seeH,seeI, seeL and seeM (Artiushin et al., 2002, Proft et al., 2003).Deletion or truncation of these genes therefore increases theimmunogenicity of the vaccine.

Other preferred genes, slaA and slab encode putative phospholipase A2toxins, which have homology with a gene recently identified in S.pyogenes (Beres et al., 2002). These are likewise believed to bevirulence factors, which exert a profound effect on the proinflammatorycascade as well as having neurotoxic, myotoxic and anticoagulantproperties.

Preferably 1, 2, 3, 4, 5, 6 or all 7 of these genes are attenuated.

Serological Discrimination

The modification of one or more genes which permit serologicaldiscrimination of the vaccine strain may be useful in differentiatingvaccinated subjects from those exposed to virulent pathogens. Thispermits the evaluation of the contribution of the vaccination to diseaseprevention and in the management of outbreaks in vaccinated populations.

A preferred target gene is that which encodes the M protein.

Vaccines based on the full length M-protein have previously shown poorefficacy in horses despite excellent immunogenicity (Sheoran et al.,2002). Truncation such as to remove regions of the IgG and\or fibrinogenbinding functional domains may further attenuate the vaccine strain (seealso Meehan et al., 2000, Meehan et al., 2001). Lack of the fibrinogenand IgG binding domains may lower the risk of induction of the immunecomplex disease purpura haemorrhagica occasionally associated withstrangles and strangles vaccines, including those based on the M-protein(Galan and Timoney 1985, Pusterla et al., 2003, Herwald et al., 2004).

Other preferred target genes encode the superantigen toxins seeH, seeI,seeL and seeM (Artiushin et al., 2002, Proft et al., 2003; see above).

Recombination Repair

Example genes may encode recombinases or other nucleic acid modifyingenzymes responsible for repair or recombination. Preferably one suchgene is partially or fully deleted such as to reduce the possibility ofstrain reversion i.e. when the above are combined with impairment of anenzyme responsible for recombination repair of the genome a liveattenuated vaccine essentially incapable of repairing the attenuatingdeletions may be achieved.

A preferred gene is the recA gene (Tao et al., 1995). Deletion of recAmay have the added advantage of increasing the sensitivity of thevaccine strain to UV light, decreasing the likelihood of environmentalpersistence.

Preferred Strains

Thus preferred strains may combine modifications (i) and (ii) with oneor more of (iii), (iv) and (v), preferably two of (iii), (iv) and (v),most preferably all three of (iii), (iv) and (v).

Preferably the strain will be engineered such that no plasmid DNA orantibiotic resistance genes remain present, such as to maintain the samesensitivity to antibiotics as the parental strain.

Preferably the Streptococcus is a Beta-haemolytic streptococcus, forexample in Lancefield group C—however as noted above Streptococcuspathogens share significant genetic identity and hence express nearidentical toxins and virulence factors.

In one embodiment the Streptococcus is S. equi which is causative ofstrangles. The present inventors have rationally selected and designed acombination of the above modifications to generate a novel liveattenuated vaccine strain known as “SHMAPR” that can be more widelyapplied throughout the equine community. This combines the followingproperties:

To reduce injection site reactions and improve safety, the sagA gene waspartially deleted. To improve the immunogenicity, the hasA gene waspartially deleted. Consequently, the train appears non-haemolytic andnon-mucoid on blood agar plates and so is readily distinguished fromwild-type strains.

It should be noted that deletion of the aroA gene (see above) wouldenable the strain to be differentiated at a genetic level from virulentS. equi (Kelly et al., 2006). However, as this gene has a near identicalhomologue in S. zooepidemicus, it cannot be utilised to develop adifferential diagnostic ELISA to rapidly determine if vaccinated animalshave seroconverted on subsequent exposure to strangles. To enableserological discrimination between vaccinated and naturally infectedhorses the M-protein was C-terminally truncated. To attenuate the strainthe genes aroB and pyrC were partially deleted although despite thispartial deletion the vaccine strain grows well in rich media. Finally,to further reduce the possibility of strain reversion the recA gene waspartially deleted.

The SHMAPR S. equi vaccine strain was attenuated when administered viathe IN, IM and subcutaneous (SC) routes in a mouse infection model. INchallenge of 30 mice, with 4×10⁶ colony forming units (cfu) of theSHMAPR strain did not cause any signs of disease (Example 3). IMchallenge of 5 mice, with 4×10⁶ colony forming units (cfu) of the SHMAPRstrain did not cause any signs of disease distal to the injection siteand injection site reactions did not exceed the mild severity limit(Example 4). SC vaccination of mice with 10⁵ or 10⁶ cfu of the SHMAPRvaccine strain was also well tolerated. All 5 mice gained weight duringthe course of the study (Example 5). The majority of IM and SCvaccinated mice developed small pustules at the injection sitecontaining live SHMAPR bacteria, indicative of immune recognition. Suchshort-term persistence of bacteria is likely to be beneficial forinduction of immunity and has previously been noted for TW 928 inhorses. Recovered bacteria maintained the non-haemolytic and acapsularphenotypes, confirming the in vivo stability of these deletions. Incontrast, mice receiving similar doses of the wild type 4047 strain viathe IN or IM routes fell ill and were all euthanased on ethical groundswithin 24 hours. In this model an IN 4047 challenge dose of 10³ cfuinduced disease in 4/5 mice within one week. Therefore, the SHMAPRvaccine strain is non-toxic and >5×10³-fold attenuated when compared tothe parental strain in mice by IN challenge.

The SHMAPR live attenuated vaccine was also found to be safe in sixponies by intranasal and intramuscular administration at doses of 1×10⁹cfu and up to 1×10⁹ cfu, respectively (Example 6). This intranasal doseof virulent S. equi 4047 has previously been shown to induce disease in96% of control ponies (Hamilton et al., 2006, Waller et al., 2007 andWaller unpublished data).

The intranasal vaccination phase proceeded without incident. Nosignificant injection site reactions were observed in any of the sixponies intramuscularly vaccinated with the SHMAPR live attenuatedvaccine.

Thus vaccines as described herein may combine stability, safety andimmunogenicity such as to permit for the possibility of intramuscularvaccination with reduced risk of harmful side effects or strainreversion. It will be appreciated that given the similarities ofstrategies of various Streptococcal pathogens, corresponding changes inother strains may likewise be expected to provide the benefits disclosedherein. Thus in another embodiment, the invention providesSHMAPR-modified vaccines based on non-S. equi strains, for example thoseshown in Table 7.

In Tables 7a and 7b homologues of certain preferred target genesdescribed herein are described.

Referring to the Tables, where sequencing of the relevant strains hasnot yet been performed (“not available”) it will be appreciated thatthose skilled in the art can nevertheless practice the present invention(in the light of the present disclosure) by identifying the homologuesby conventional methods—for example PCR, blotting or the like (see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al, 1989). The identification of such genes does not per se form partof the invention.

Referring to the Tables, if a homolog of a gene is believed not to bepresent in the relevant strain (“none”) then it will be appreciated thatmodification of that gene in that strain is not required by the presentinvention.

Other particular preferred strains include also those strains shown inTable 8.

In one aspect, the invention provides S. equi strain SHMAPR, asdeposited at the National Collection of Type Cultures (NCTC), HealthProtection Agency, Centre for Infections 61, Colindale Avenue, LONDONNW9 5EQ, United Kingdom under accession number: 13412.

Thus in all aspects and embodiments described herein (e.g. strains,methods, processes, vaccines) this is optionally a preferred strain.

The invention further relates to a microbiological pure culturecomprising bacteria according to the deposited strain. It goes withoutsaying that next generations of bacteria from the deposited strain arealso included.

The culture can e.g. be obtained by growing said bacteria at atemperature between 30 and 41° C. Bacteria can be grown e.g. in ToddHewitt medium.

Production of Vaccines

The invention further provides a process for producing a Streptococcusvaccine strain, which process comprises:

(a) making the following modifications to its genome:

-   -   (i) attenuation of one or more essential biosynthetic genes,    -   (ii) attenuation of one or more genes which encode a haemolytic        toxin, or protein involved in the production thereof, plus        preferably any one, two, or most preferably three of the        following modifications:    -   (iii) attenuation of one or more genes which encode a protein        responsible for reduced immunogenicity,    -   (iv) modification of one or more genes such as to permit        serological discrimination of the vaccine strain based on        analysis of a protein encoded by said genes, and    -   (v) attenuation of one or more genes which encode an enzyme        responsible for recombination repair of the genome.        (b) optionally culturing the resulting strain.

The choice of genes may include any of those described above. As notedabove, well-defined and deliberately made mutations involving thedeletion of fragments of the gene or even the whole gene or theinsertion of heterologous DNA-fragments or both, have the advantage, incomparison to classically induced mutations, that they will not readilyrevert to the wild-type situation. Thus, in one embodiment, theattenuation is deletion of at least 100 nucleotides. In one embodimentthe deletion corresponds to that shown in any of Sequence Annexes 1-6.Optionally the primers described in the Examples hereinafter may be usedin the preparation of deletions. These primers and their use in theprocess of this aspect thus form another aspect of the invention.

The vaccine strain (optionally obtained from culturing as describedabove, or from the relevant depositary institution) may be used in aprocess to prepare a vaccine, whereby it is combined with apharmaceutically acceptable carrier.

The vaccine may be freeze dried, as described in more detail below.

The invention further provides a vaccine for vaccination against aStreptococcal pathogen as described herein comprising live bacteria ofany Streptococcus attenuated vaccine strain described above and apharmaceutically acceptable carrier. Such a carrier may be as simple aswater, but it may e.g. also comprise culture fluid in which the bacteriawere cultured. Another suitable carrier is e.g. a solution ofphysiological salt concentration.

Methods and Uses of Vaccines

In all vaccines, methods and uses described herein the vaccine ispreferably a live vaccine. However use of inactivated (killed) vaccineformulations is also embraced by the present invention, should that bepreferred by the user.

The invention further provides a method for immunising a subject againsta Streptococcal pathogen, which method comprises administering to saidsubject the respective Streptococcus attenuated vaccine strain describedabove in sufficient amount to raise a protective immune responsethereto.

“Respective” in this context means either the Streptococcal pathogenwhich was attenuated to produce the vaccine strain, or a differentpathogen which is sufficiently immunologically cross reactive such thatthe vaccine strain provides protection therefrom.

For example in one embodiment an S. equi vaccine strain may be used forcombating Streptococcus equi or Streptococcus zooepidemicus infection inhorses.

Preferably the method is for vaccination against a disease shown inTable 7, by use of the appropriate live vaccine strain preparedaccording to the present invention.

It will be appreciated by those skilled in the art that immunisation, orprotection, in this context does not necessarily circumscribe completesuccess (i.e. that a subject so vaccinated would never be susceptible toinfection by said pathogen). Rather it is used in its art-recognisedsense to be a prophylactic measure with the aim of mitigating theseverity of, or reducing the incidence of, said infection.

Strains described herein may be “avirulent” by which it would beunderstood not to be able to cause the relevant disease in the subjectand includes those which a person of skill in the art would considersafe for administering to the subject as a vaccine. For example, in thecontext of a strangles vaccine, a strain causing minor clinical signs,including fever, serous or mucopurulent nasal discharge or oculardischarge, is within the scope of the present invention since suchclinical signs are considered acceptable vaccine side effects.

In other aspects the invention provides:

Use of Streptococcus attenuated vaccine strain described above insufficient amount to raise an immune response thereto in a method forimmunising a subject against the respective Streptococcus pathogen;

A Streptococcus attenuated vaccine strain described above for use in amethod for immunising a subject against the respective Streptococcuspathogen;

Use of Streptococcus attenuated vaccine strain described above in thepreparation of a medicament for immunising a subject against therespective Streptococcus pathogen. The medicament may comprise asufficient amount or dose to raise an immune response thereto in thesubject.

Modes of Administration and Dosage

A vaccine according to the present invention can be administered invarious forms. It can e.g. be administered parenterally, e.g.intramuscularly, subcutaneously or intradermally, it can also be givenorally, submucosally (e.g. in the lip) or it can be given intranasally.

In respect of strangles, the nasal mucosa is the most common ported'entree for Streptococcus equi infection. Therefore, the nose is themost natural place for the application of the live attenuated vaccineaccording to the invention. In addition, this application site has theadvantage that it is easily reached, and that the vaccine can e.g. beadministered by spraying. Thus, in one preferred form, the vaccine ofthe present invention is suitable for intranasal application. Othervaccines are commonly administered via the intramuscular route. TheSHMAPR live attenuated vaccine has been specifically designed such thatthis route can be utilised. Therefore, in another preferred form, thevaccine of the present invention is suitable for intramuscularapplication. Due to its attenuated characteristics, the vaccine can beused to protect horses at any age, including young horses e.g. between 6and 12 months of age.

A vaccine according to the present invention may comprise any dose ofbacteria, sufficient to evoke an immune response. Doses ranging between10³ and 10⁹ bacteria are e.g. very suitable doses.

A vaccine according to the present invention may comprise any volume ofbacteria, suitable for the route of administration selected. Volumesranging between 0.2 ml and 2 ml bacteria are e.g. very suitable forintramuscular administration. Volumes ranging between 2 ml and 20 mlbacteria are e.g. very suitable for intranasal administration.

A vaccine according to the present invention may be administered using avariety of different dosing regimens, sufficient to evoke an immuneresponse. Doses administered between 2 and 12 weeks are e.g. verysuitable for naïve animals. Doses administered between 12 and 52 weeksare e.g. very suitable for primed animals.

Storage

There are several ways to store live organisms. Storage in arefrigerator is e.g. a well-known method. Also often used is storage at−70° C. in a buffer containing glycerol. Bacteria can also be kept inliquid nitrogen. Freeze-drying is another way of conservation.Freeze-dried bacteria can be stored and kept viable for many years.Storage temperatures for freeze-dried bacteria may well be above zerodegrees, without being detrimental to the viability. Freeze-drying canbe done according to all well-known standard freeze-drying procedures.

Optional beneficial additives, such as e.g. skimmed milk, trehalose,gelatin or bovine serum albumin can be added in the freeze-dryingprocess. Therefore, in a more preferred form, the vaccine is in afreeze-dried form.

Other Vaccine Constituents

In another embodiment, the vaccine of the present invention additionallycomprises another attenuated pathogen or antigenic material from anotherpathogen. Such a pathogen may e.g. be another bacterium or a parasite.Also it can be of viral origin. Usually, the other pathogen or antigenicmaterial thereof will be a horse pathogen. A vaccine according to theinvention that also comprises such an additional attenuated pathogen orantigenic material from another pathogen has the advantage that itinduces protection against several infections at the same time. Horsepathogens or antigenic material thereof that can advantageously be addedare e.g. Potomac fever agent, Rhodococcus equi, Clostridium botulinumClostridium tetanii, Burkholdiera mallei, Streptococcus zooepidemicus,Vesicular Stomatitis Virus, Borna disease virus, Equine influenza virus,African horse sickness virus, Equine arteritis virus, Equine herpesvirus1-4, Infectious anemia virus, Equine encephalomyelitis viruses (Eastern,Western and Venezuelan), West Nile virus, Rabies virus, Hendra diseasevirus and Japanese B encephalomyelitis virus.

The vaccine may also comprise an adjuvant. Adjuvants are non-specificstimulators of the immune system. They enhance the immune response ofthe host to the invading pathogen. Examples of adjuvants known in theart are Freunds Complete and Incomplete adjuvant, vitamin E, non-ionicblock polymers, muramyldipeptides, ISCOMs (immune stimulating complexes,cf. for instance EP 109942), Quill A, mineral oil, vegetable oil, andCarbopol (a homopolymer).

Adjuvants, specially suitable for mucosal application are e.g. the E.coli heat-labile toxin (LT) or Cholera toxin (CT).

In addition, the vaccine may comprise one or more stabilisers. Also, thevaccine may comprise one or more suitable emulsifiers, e.g. Span orTween.

Thus in all aspects and embodiments described herein (e.g. strains,methods, processes, vaccines) the following may optionally be includedalong with the vaccine strain:

a) Stabilising proteins to facilitate freeze-drying e.g. any describedabove.b) Adjuvant e.g. selected from the group consisting of E. coliheat-labile toxin and Cholera toxin.c) Other attenuated pathogen or antigenic material from anotherpathogen, as appropriate to the subject to be treated (i.e. to product amultivalent vaccine). For example, a multivalent vaccine suitable foruse in horses may include attenuated pathogen or antigenic materialtherefrom, from the group consisting of Potomac fever agent, Rhodococcusequi, Clostridium botulinum Clostridium tetanii, Burkholdiera mallei,Streptococcus zooepidemicus, Vesicular Stomatitis Virus, Borna diseasevirus, Equine influenza virus, African horse sickness virus, Equinearteritis virus, Equine herpesvirus 1-4, Infectious anemia virus, Equineencephalomyelitis viruses (Eastern, Western and Venezuelan), West Nilevirus, Rabies virus, Hendra disease virus and Japanese Bencephalomyelitis virus.

The resulting strains, methods, processes, vaccines etc. including oneor more of these things form other aspects of the invention.

Any sub-titles herein are included for convenience only, and are not tobe construed as limiting the disclosure in any way.

The invention will now be further described with reference to thefollowing non-limiting Figures and Examples. Other embodiments of theinvention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may beused by those skilled in the art to carry out the invention, is herebyspecifically incorporated herein by cross-reference.

FIGURES

FIG. 1: Relative location of the primers described in the Examplesbelow.

FIG. 2: Mean % change in mass following intranasal challenge with S.equi 4047 or SHMAPR. Error bars indicate 95% confidence intervals.

FIG. 3: Mean % change in mass following intramuscular challenge with S.equi 4047 or SHMAPR. Error bars indicate 95% confidence intervals.

FIG. 4: Mean % change in mass following subcutaneous challenge with1×10⁶ or 1×10⁵ cfu of S. equi SHMAPR. Error bars indicate 95% confidenceintervals.

FIG. 5: Mean pathology score in control and SHMAPR challenged ponies.Control data represents the mean of 4 independent challenges with S.equi 4047 in a total of 23 ponies (Hamilton et al., 2006, Waller et al.,2007 and Robinson, unpublished data). Error bars represent the 95%confidence interval.

FIG. 6: Mean rectal temperature. Error bars indicate 95% confidenceinterval.

FIG. 7: Mean pathology score. Error bars indicate 95% confidenceinterval.

SEQUENCE ANNEXES 1-6

Sequence Annex 1—sagA deletion:Sequence Annex 2—hasA deletion:Sequence Annex 3—seM deletion:Sequence Annex 4—aroA deletion:Sequence Annex 5—pyrC deletion:Sequence Annex 6—recA deletion:

EXAMPLES Example 1 Selection of a Mutant Strain

An example vaccine was derived from a field isolate of Streptococcusequi responsible for causing strangles in a New Forest pony in Hampshirein 1990 and is the subject of the Streptococcus equi genome-sequencingproject (http://www.sanger.ac.uk/Projects/S_equi/). The field strain wasdesignated strain 4047. This strain was grown overnight, aerobically at37° C., on blood agar and then inoculated in Todd Hewitt medium andsubjected to well-described DNA mutation techniques (Maguin et al.,1992) to achieve the desired vaccine characteristics.

Briefly, the partial deletion of each target gene was performed asfollows:

Upstream and downstream pieces of DNA homologous to the target gene werecloned into the pGhost9 plasmid via EcoRI and SalI restriction sites tocreate a copy of the target gene lacking the desired internal portion ofthe coding sequence. Details of the primers used to generate therequired plasmids, the section of the genes to be deleted and aschematic for primer use can be found in Tables 1 and 2 and FIG. 1.

pGhost9 plasmid containing the desired homologous DNA was thentransformed into competent Streptococcus equi strain 4047 viaelectroporation and strains containing the plasmids were grown for 48hours at 28° C. on Todd Hewitt medium with 0.5 μg of erythromycin perml. An overnight (16 h) culture was diluted 100-fold in Todd Hewittmedium containing erythromycin and incubated at 28° C. until on OD₆₀₀ of0.3 was reached (mid exponential phase). The culture was then shifted to37.5° C. for 150 min to initiate integration of the plasmid in to thechromosome by homologous recombination, as the plasmid can not replicateat 37° C.

Samples were diluted and plated at 37° C. on Todd Hewitt agar containing0.5 μg of erythromycin per ml (to identify insertion mutants) andwithout erythromycin (to determine the viable cell count). Integrationof the plasmid into the chromosome was confirmed by PCR.

To excise the inserted vector and generate the desired gene deletion,insertion mutants were grown overnight in Todd Hewitt medium (withouterythromycin) at 37° C. The culture was then diluted 10³-fold in ToddHewitt medium and incubated at 28° C. without erythromycin, untilstationary phase (about 18 h); this step stimulates recombination byallowing plasmid replication. Cultures were then diluted and platedwithout erythromycin at 37° C., to allow loss of the excised plasmid.Colonies were transferred with toothpicks to selective (containing 0.5μg of erythromycin per ml) and non-selective plates. Colonies in whichexcision had occurred were phenotypically erythromycin sensitive.

Deletion mutant strains were identified by PCR across the desireddeletion site. In such experiments a smaller target gene DNA fragmentwas amplified from the desired deletion mutant strain when compared withthe parental Streptococcus equi 4047 strain (Table 2).

Each gene was deleted sequentially in the order sagA, hasA, seM, aroB,pyrC and recA. recA was deleted last as the deletion process requiresthe presence of the recA gene product.

A strain with deletions in the genes sagA, hasA, seM, aroB, pyrC andrecA was selected and designated SHMAPR. PCR was performed across eachof the deleted target genes using the primers in Table 1 and theproducts purified and sequenced on an ABI3100 automated sequencer usingwell-described protocols to confirm that the desired deletions had beengenerated. Details of each deletion generated are presented in Table 2.

Owing to deletion of part of the sagA gene, the SHMAPR strain appearsnon-haemolytic on blood agar plates.

Owing to deletion of part of the hasA gene, the SHMAPR strain appearsnon-mucoid on agar plates and sediments in liquid culture.

TABLE 1 Target gene Primer name Primer sequence sagA 5′sagA¹GGGGAATTCTGAGGTACTAGCCATCTGTC sagA NDEL²GGGAAGCTTAGCAAATTGTAACATAATGCTTACC sagA CDEL³GGGAAGCTTGCTGAGCCAAAAGCGTAAAC 3′sagA⁴ GGGGTCGACAAAACTCAGCCACACTGGTC hasA5′hasA2¹ GGGGAATTCAAGGGAAGGGCTGGGCAATATAAGG hasA NDEL²GGGGATATCATTTCTGACATTAAGGTGACCCGTC hasA CDEL³GGGGATATCTGGAACAAGTCCTTCTTTAGAGAG 3′hasA2⁴GGGGTCGACAGGGCTGTAGGACAAACAAATGCAG seM 5′SEM stop¹GGGGAATTCATGTTTTAGAGAAATAACAAGC SEM NDEL stop²GGGGATATCATTTTACATCGATGAAAGGTG SEM CDEL stop³GGGGATATCTGAGATGCTAAGGTAGCAGAGC 3′SEM CENT⁴GGGGTCGACGTTTTCTTTGCGTTTAGGAGACACC aroB ASW31¹GACGAATTCTGTCTGAAAGGCAGCTAGAG ASW32² GACGACGATATCGGATAGTCATTGATACGAGACASW33³ GCTAGATATCGCCTGAGAAGGCT ASW34⁴ GACGACGTCGACTGGTAAGACCTGGACAACAGZM24⁵ ACACCTGATCTTGCCTTGTC pyrC ASW35¹ GACGAATTCGCAGCAGATATTGGAGTAAGGASW36² GACGACAAGCTTGCCACCTGATCTAGCTGTGAT ASW37³GACGACAAGCTTAGCGTTTGGTAACAGAAGCC ASW38⁴ GACGACGTCGACTACGTTTCGGATTCTTGGGCZM23⁵ GGCAGGCTATTATGGCTAAG recA ASW57¹ GACGACGAATTCTTATTGCTTGCTAGTCAGCCASW58² GACGACGATATCAAGGCTGCAATACCACCTTC ASW59³GACGACGATATCGAAGGCATCTCACGTACAGG ASW60⁴ GACGACGTCGACTTGACGATCGCTGTTAAGCC⁵Additional primer used for sequencing. Restriction sites used forcloning are underlined

TABLE 2 Size of Size of Target deleted 4047 strain Gene DeletionDeletion gene PCR product PCR product size generated size sagA 945 bp1071 bp  165 bp 16 bp to 126 bp 141 bp hasA 722 bp 1033 bp 1254 bp 577bp to 311 bp 888 bp seM 810 bp 1620 bp 1605 bp 349 bp to 810 bp 1158 bparoB 1243 bp  2270 bp 1083 bp 46 bp to 1027 bp  1073 bp pyrC 1387 bp 2472 bp 1413 bp 204 bp to 1085 bp  1288 bp recA 731 bp 1257 bp 1152 bp330 bp to 526 bp 855 bp

The SHMAPR vaccine strain was then tested for its attenuated characteras described in the Examples below.

Example 2 Preparation of Vaccine

Streptococcus equi strain SHMAPR and the wild type parent 4047 strainwere grown overnight, aerobically at 37 degree. C., on blood agar andthen inoculated in Todd Hewitt medium containing 10% foetal calf serum.For the vaccination/challenge studies, the strains were cultured for 6hours at 37 degree. C. in 100 ml of Todd Hewitt medium containing 10%foetal calf serum to an OD_(600nm) of 0.3. At this density the viablenumber of S. equi is 2×10⁸ cfu/ml.

Example 3 Intranasal Safety Test of the Vaccine Strain SHMAPR in Mice

In this example, the rate of attenuation of the S. equi SHMAPR ascompared to the wild-type strain 4047 has been tested in mice. 4×10⁸ CFUof the mutant strain as well as the parent 4047 wild-type strain wereapplied intranasally to mice and mortality was recorded.

Animals

BALB/c mice, 4 weeks of age, obtained from Charles River Ltd, were usedfor the experiment.

Vaccination/Challenge Cultures

Streptococcus equi strain SHMAPR and the wild type parent 4047 strainwere grown overnight, aerobically at 37° C., on blood agar and theninoculated in Todd Hewitt medium containing 10% foetal calf serum. Thestrains were then cultured for 6 hours at 37° C. in 20 ml of Todd Hewittmedium containing 10% foetal calf serum to an OD_(600nm) of 0.3. At thisdensity the viable number of S. equi is 2×10⁸ cfu/ml.

Treatment

At 4 weeks of age, 1 group of 30 mice sedated with 100 mg/kg ketaset,was challenged intranasally (20 μl) with 4×10⁶ cfu S. equi strain 4047and 1 group of 30 mice sedated with 100 mg/kg ketaset, was treatedintranasally with 4×10⁶ cfu S. equi strain SHMAPR.

After the treatments, clinical signs of disease, weight loss andmortality was recorded for 5 days. Histopathological examination of allmice was used to determine the extent of disease progression.

Results:

The results after intranasal challenge of 4 weeks old mice with 4×10⁸cfu strain 4047 or strain SHMAPR are shown in Table 3.

A severe infection in 28/30 mice challenged with the 4047 strain wasobserved within 24 hours. All mice in the wild type challenge groupswere humanely euthanased at or before 24 hours after initial challenge.On post mortem examination, it was apparent that these mice had diedfrom pneumonia and septicaemia rather than the more classical clinicalcourse of strangles.

None of the 30 mice challenged with an identical dose of S. equi SHMAPRshowed any clinical signs of disease. All mice continued to gain weight(FIG. 2). Histopathological examination of all mice 5 days postinfection identified only mild signs of infection in 6/30 mice.

TABLE 3 Number Clinical Histopa- of signs of thological Group mice RouteDose Mortality disease disease 4047 30 IN 4 × 10⁶ 30 28 29 (on day 1)SHMAPR 30 IN 4 × 10⁶  0 0 6 (by day 5)

Conclusions

Other experiments following the same methods for strain preparation andadministration have determined that an intranasal dose of 1×10³ cfu ofS. equi 4047 was sufficient to induce clinical disease in 4 of 5 mice at5 days post challenge (Waller, unpublished data). Therefore, the SHMAPRstrain is over 5×10³-fold attenuated in the intranasal mouse challengemodel. The mild histological signs identified in 6/30 SHMAPR challengedmice indicates that this strain can stimulate an immune response inmice.

Example 4 Intramuscular Safety Test of the Vaccine Strain SHMAPR in Mice

In this example, the rate of attenuation of the S. equi SHMAPR ascompared to the wild-type strain 4047 has been tested in mice. 4×10⁶ CFUof the mutant strain as well as the parent 4047 wild-type strain wereapplied intramuscularly into the left leg of mice and mortality wasrecorded.

Animals

BALB/c mice, 4 weeks of age, obtained from Charles River Ltd, were usedfor the experiment.

Vaccination/Challenge Cultures

Streptococcus equi strain SHMAPR and the wild type parent 4047 strainwere grown overnight, aerobically at 37° C., on blood agar and theninoculated in Todd Hewitt medium containing 10% foetal calf serum. Thestrains were then cultured for 6 hours at 37° C. in 20 ml of Todd Hewittmedium containing 10% foetal calf serum to an OD_(600nm) of 0.3. At thisdensity the viable number of S. equi is 2×10⁸ cfu/ml.

Treatment

At 4 weeks of age, 1 group of 5 mice was challenged intramuscularly (20μl) with 4×10⁸ cfu S. equi strain 4047 and 1 group of 5 mice waschallenged intramuscularly (20 NI) with 4×10⁸ cfu S. equi strain SHMAPR.

After the treatments, clinical signs of disease, weight loss andmortality was recorded for 5 days. Histopathological examination of allmice was used to determine the extent of disease progression.

Results:

The results after intramuscular challenge of 4 weeks old mice with 4×10⁸cfu strain 4047 or strain SHMAPR are shown in Table 4.

On day 1, all 5 mice injected IM with S. equi 4047 had severe swellingof their left leg and had ruffled coats. All of these mice looked veryill and were euthanased. All 5 mice injected with the SHMAPR straindeveloped swollen left legs, but remained active throughout the study.By day 5 one mouse had resolved its mild injection site reaction. All S.equi SHMAPR challenged mice gained weight as normal during the studyperiod (FIG. 3).

On post mortem examination, 1 mouse challenged with S. equi 4047 haddeveloped histological disease away from the injection site. None of the5 mice challenged with an identical dose of S. equi SHMAPR hadhistological signs distal to the injection site. 4 SHMAPR vaccinatedmice had small injection site pustules 5 days post challenge, from whichthe original S. equi SHMAPR strain was isolated, highlighting that theSHMAPR strain can persist for a short time at the injection site.

TABLE 4 Number Clinical Histopa- of signs of thological Group Route Dosemice Mortality disease disease 4047 IM 4 × 10⁶ 5 5 5 1 (on day 1) SHMAPRIM 4 × 10⁶ 5 0 0 0 (by day 5)

Conclusions

The S. equi SHMAPR strain produced dramatically less disease andinjection site swelling than S. equi 4047 when injected intramuscularly.The SHMAPR strain could persist at the injection site for a short periodof time, which may enhance the stimulation of the immune system.Injection site reactions in mice did not cause any clinical signs ofdisease and mice continued to gain weight normally during the studyperiod. Therefore, the SHMAPR live attenuated vaccine appears ideallysuited for intramuscular administration.

Example 5 Subcutaneous Safety Test of the Vaccine Strain SHMAPR in Mice

In this example, the safety of two doses of the S. equi SHMAPR wastested in mice. 1×10⁶ or 1×10⁵ CFU of the mutant strain were appliedsubcutaneously into the neck scruff of mice and mortality was recorded.No mice were challenged subcutaneously with parental strain 4047 becauseof the severe disease observed in earlier intranasal and intramuscularchallenges with this strain.

Animals

BALB/c mice, 4 weeks of age, obtained from Charles River Ltd, were usedfor the experiment.

Vaccination/Challenge Cultures

Streptococcus equi strain SHMAPR was grown overnight, aerobically at 37°C., on blood agar and then inoculated in Todd Hewitt medium containing10% foetal calf serum. The strain was then cultured for 6 hours at 37°C. in 20 ml of Todd Hewitt medium containing 10% foetal calf serum to anOD_(600nm) of 0.3. At this density the viable number of S. equi is 2×10⁸cfu/ml.

Treatment

At 4 weeks of age, 1 group of 5 mice was challenged subcutaneously (10μl) with 1×10⁶ cfu S. equi strain SHMAPR and 1 group of 5 mice waschallenged subcutaneously (10 μl) with 1×10⁵ cfu S. equi strain SHMAPR.

After the treatments, clinical signs of disease, weight loss andmortality was recorded for 5 days. Histopathological examination of allmice was used to determine the extent of disease progression.

Results:

The results after subcutaneously challenge of 4 weeks old mice with1×10⁶ cfu or 1×10⁵ cfu of strain SHMAPR are shown in Table 5.

All mice showed signs of injection site reactions, which did not exceedthe mild severity limit. The injection site reaction in one mousechallenged with 1×10⁵ cfu of SHMAPR resolved by day 7 post-challenge.All mice remained active and gained weight normally during the studyperiod (FIG. 4).

On post mortem examination, none of the mice had histological signsdistal to the injection site. 5/5 mice challenged subcutaneously with1×10⁶ cfu and 4/5 mice challenged subcutaneously with 1×10⁵ cfu S. equiSHMAPR had small injection site pustules 7 days post challenge, fromwhich the original S. equi SHMAPR strain was isolated, highlighting thatthe SHMAPR strain can persist for a short time at the injection site.

TABLE 5 Number Clinical Histopa- of signs of thological Group Route Dosemice Mortality disease disease SHMAPR SC 1 × 10⁶ 5 0 0 0 SHMAPR SC 1 ×10⁵ 5 0 0 0

Conclusions

The S. equi SHMAPR strain was well tolerated by the subcutaneous route.All mice remained active and healthy throughout the study period. TheSHMAPR strain could persist at the injection site for a short period oftime, which may enhance the stimulation of the immune system. Injectionsite reactions in mice did not cause any clinical signs of disease andmice continued to gain weight normally during the study period.Differences in the % change in mass between the groups only becamesignificant on day 7 and could be due to mouse/mouse variation given thesmall group sizes used. Therefore, the SHMAPR live attenuated vaccinealso appears ideally suited for subcutaneous administration.

Example 6 Intranasal and Intramuscular Safety Test of the Vaccine StrainSHMAPR in Welsh Mountain Ponies

In this example, the safety and immunogenicity of the SHMAPR vaccinestrain in horses by intranasal and intramuscular vaccination wasdetermined.

Animals

Six male Welsh Mountain Ponies supplied by Mr. R. Beedles, Shropshire,UK were used. Ponies were approximately 8 months of age at the time ofthe first vaccination.

Vaccination Cultures

Streptococcus equi strain SHMAPR and the wild type parent 4047 strainwere grown overnight, aerobically at 37° C., on blood agar and theninoculated in Todd Hewitt medium containing 10% foetal calf serum. Thestrains were then cultured for 6 hours at 37° C. in 20 ml of Todd Hewittmedium containing 10% foetal calf serum to an OD_(600nm) of 0.3. At thisdensity the viable number of S. equi is approximately 2×10⁸ cfu/ml.

Treatment

On day 0 the ponies received an intranasal dose of 1×10⁸ cfu SHMAPR.This intranasal dose of virulent S. equi 4047 has previously been shownto induce disease in 96% of control ponies (Hamilton et al., 2006;Waller et al., 2007; Waller unpublished data).

On day 14 the ponies received an intramuscular dose of 1×10⁹ cfu, 1×10⁸cfu or 1×10⁷ cfu SHMAPR into the right side of their neck according toTable 6:

TABLE 6 Number of ponies/ID Intranasal Intramuscular Group numbersvaccine dose vaccine dose A 2/4277, 4530 1 × 10⁸ cfu 1 × 10⁹ cfu B2/5066, 5251 1 × 10⁸ cfu 1 × 10⁸ cfu C 2/5756, 5793 1 × 10⁸ cfu 1 × 10⁷cfu

Results:

The SHMAPR live attenuated vaccine was also found to be safe in sixponies by intranasal and intramuscular administration at doses of 1×10⁸cfu and up to 1×10⁹ cfu, respectively.

The intranasal vaccination phase proceeded without incident. No increasein the size of submandibular lymph nodes or signs of mucopurulent nasaldischarge were observed during this two week period.

Ponies were then intramuscularly vaccinated with 1×10⁹ cfu, 1×10⁸ cfu or1×10⁷ cfu of the SHMAPR live attenuated vaccine. No significantinjection site reactions were observed in any of the vaccinated poniesand all ponies had normal neck movements and appetites, indicating thatthe SHMAPR vaccine can be safely administered via this vaccinationroute.

Following post mortem examination a small pustule containing the SHMAPRlive attenuated vaccine strain was found in one pony vaccinated with1×10⁸ cfu of the SHMAPR live attenuated vaccine. The pustule did notappear to be progressing beyond the injection site. This type of lesionmay be highly advantageous in stimulating a prolonged protective immuneresponse against infection with a field strain. There was no evidencefor spread of the SHMAPR strain to draining lymph nodes. These datademonstrate that the SHMAPR strain resulted in a much reducedintramuscular injection site lesion compared with published data usingthe Intervet live attenuated strain administered by the intramuscularroute (Jacobs et al., 2000).

Conclusions

The SHMAPR live vaccine strain is safe in ponies for administration bythe intranasal and intramuscular routes.

Example 7 Efficacy of the Vaccine Strain SHMAPR in Welsh Mountain Ponies

In this example, the efficacy of the SHMAPR vaccine strain in horses byintramuscular vaccination was determined after challenge with virulentS. equi.

Animals

Eighteen Welsh Mountain Ponies were used. Ponies were approximately 14months of age at the time of the first vaccination.

Vaccination Cultures

Streptococcus equi strain SHMAPR and the wild type parent 4047 strainwere grown overnight, aerobically at 37 degree C., on blood agar andthen inoculated in Todd Hewitt medium containing 10% foetal calf serum.The strains were then cultured for 6 hours at 37 degree C. in 20 ml ofTodd Hewitt medium containing 10% foetal calf serum to an OD_(600nm) of0.3. At this density the viable number of S. equi is approximately 2×10⁸cfu/ml. For vaccination, the required dose of SHMAPR was centrifuged for10 minutes at 3000 rpm and resuspended in the appropriate volume ofpre-warmed and gassed Todd Hewitt medium containing 10% foetal calfserum.

Treatment

On day 0 9 ponies received an intramuscular dose of 1×10⁸ cfu SHMAPR in200 μl Todd Hewitt medium containing 10% foetal calf serum. This dose ofSHMAPR was previously shown to be safe over a two-week follow-up periodvia the intramuscular route (see example 6). The remaining 9 ponies werevaccinated with an equivalent volume of Todd Hewitt medium containing10% foetal calf serum.

Ponies were monitored for 8 weeks and then a second vaccination wasadministered as above.

Ponies were monitored for a further 8 weeks and then challenged viaadministration of a dose of 1×10⁸ cfu of virulent S. equi strain 4047.This dose has previously been shown to induce disease in >96% of horses.

Ponies were followed for signs of disease over a three week period. Allponies were then euthanased and subjected to post mortem examination.The number of abscesses formed in the lymph nodes of ponies were notedand the amount of disease in the vaccinated vs. control populationscalculated.

Results:

Following the first vaccination, the SHMAPR live attenuated vaccine wasfound to slowly induce local injection site reactions in 4 of 9 poniesvaccinated. Earliest indications occurred two to three weeks postvaccination. Lesions were generally painless and did not result in lossof appetite, but were unsightly. Drainage of lesions was performed by aveterinary surgeon following sedation of affected ponies. The lesions inall four ponies then rapidly resolved. However, lesions in two of theseponies had not fully resolved at the time of the administration of thesecond vaccination and these ponies were not revaccinated. Both ponieshad fully recovered shortly afterwards and were included in thechallenge phase of the study.

Following the administration of the second vaccination to the remaining7 ponies, slow forming injection site lesions occurred in one pony. Thispony had been unaffected following the first vaccination. This lesionwas drained; the pony made a full recovery and was included in thechallenge phase of this study.

Following challenge with virulent S. equi 8 of 9 control poniesdeveloped pyrexia over the 3 week observation period (mean number ofdays pyrexic=4.0). In contrast only one vaccinated pony developedpyrexia over the same period (mean number of days pyrexic=0.1) (FIG. 6).Ponies were euthanased as it became obvious that they had developeddisease and before the rupture of lymph node abscesses on ethicalgrounds. The first control pony was euthanased 11 days post challengeand only 2 control ponies reached the endpoint of the study 18 days postchallenge. All 9 vaccinated ponies reached the end of the study.

On post mortem examination abscesses were found in the retropharyngeallymph nodes of all control ponies but only 2 of the 9 vaccinated ponies.One of these 2 vaccinated ponies had only received one vaccination. Themean pathology score for controls was 33.3 (±6.3) compared with a meanof 5.0 (±4.6) for vaccinated ponies (FIG. 7).

Conclusions

The SHMAPR live vaccine strain is efficacious for the prevention ofstrangles.

TABLE 7A most preferred genes and homologues Bacterium Disease inanimals Disease in humans sagA hasA S. equi Strangles in horses. 0 0(beta-haemolytic) S. zooepidemicus Abortion, mastitis, keratitis,Nephritis, meningitis Currently on Currently on (beta-haemolytic) woundinfections, abscesses contig contig Sequence at in a wide variety ofanimals. zoo32c02.p1k GZOO 1414 http://www.sanger.ac.uk/ Residues203925- 1396- cgi-bin/blast/submitblast/ 204086 1a02.w2k1396s_zooepidemicus incomplete residues 4614- last accessed on Jul. 01, 20085864 S. pyogenes None. Necrotising facititis, Residues 171838- Residues1821324- (beta-haemolytic) pharyngitis, sepsis. 172932 1822563 Sequenceat http://www.sanger.ac.uk/ Projects/S_pyogenes/ S. uberis Mastitis incattle. NONE Residues 1676849- http://www.sanger.ac.uk/ 1678089Projects/S_uberis/ S. pneumoniae Pneumonia in horses. Pneumonia NONENONE http://www.sanger.ac.uk/ Projects/S_pneumoniae/23F/ S. agalactieaeMastitis in cattle Neonatal meningitis, NONE NONE NC_007432 toxic shock,skin and soft tissue infections, bacteraemia S. suis Meningitis,septicemia, Meningitis, endocarditis NONE NONE http://www.sanger.ac.uk/arthritis, bronchopneumonia, spondylodiscitis, Projects/S_suis/endocarditis, encephalitis, Streptococcal toxic shock abortions andabscesses in pigs. syndrome, sepsis, arthritis, endophtalmitis,pneumonia S. iniae Meningiitis, panophthalmitis Bacteraemic cellulitisContig 00719 Contig 00200 (beta-haemolytic) in fish Subcutaneous abscessresidues 19433- Residues 24289- http://www.hgsc.bcm.tmc.edu/ in dolphins19795 25257 bcm/blast/microbialblast.cgi Last searched on Jul. 25, 2007Bacterium SeM aroB pyrC recA S. equi 0 0 0 0 (beta-haemolytic) S.zooepidemicus Currently on Currently on Currently on Currently on(beta-haemolytic) contig contig contig contig Sequence at GZOO 1414J25443Df01.p1k J25443Df01.p1k zoo122a01.p1k http://www.sanger.ac.uk/1396- Residues 384986- Residues 11359- residues 171841-cgi-bin/blast/submitblast/ 1a02.w2k1396 386020 12639 172932s_zooepidemicus incomplete residues 29648- last accessed on Jul. 01,2008 31387 S. pyogenes Residues 226656- Residues 582244- Residues1097957- Residues 1756634- (beta-haemolytic) 227440 583280 10992411757727 Sequence at http://www.sanger.ac.uk/ Projects/S_pyogenes/ S.uberis Residues 147388- NONE Residues 995140- Residues 1764212-http://www.sanger.ac.uk/ 148092 996400 1765308 Projects/S_uberis/ S.pneumoniae NONE Residues 1312069- Residues 1048493- Residues 1901125-http://www.sanger.ac.uk/ 1312660 1049757 1902171Projects/S_pneumoniae/23F/ S. agalactieae ref|YP_329152.1|ref|YP_330019.1| ref|YP_329756.1| ref|YP_330622.1| NC_007432 S. suisNONE NONE Residues 888290- 67730-68754 http://www.sanger.ac.uk/ 889588Projects/S_suis/ S. iniae NONE NONE NONE contig 00203 (beta-haemolytic)residues 18147- http://www.hgsc.bcm.tmc.edu/ 19153bcm/blast/microbialblast.cgi Last searched on Jul. 25, 2007

TABLE 7B other preferred genes and homologues Bacterium Disease inanimals Disease in humans slaA seeI S. equi Strangles in horses. None 00 (beta-haemolytic) Also has second homologue virtually identical to theone found in zooepidemicus 5.7e−105 824084-824656 S. zooepidemicusAbortion, mastitis, keratitis, Nephritis, meningitis Contig NONE(beta-haemolytic) wound infections, abscesses zoo122a01.p1k Sequence atin a wide variety of animals. Residues 175312- http://www.sanger.ac.uk/175881 cgi-bin/blast/submitblast/ s_zooepidemicus S. pyogenes None.Necrotising facititis, NONE in Manfredo 6.6e−167 (beta-haemolytic)pharyngitis, sepsis. This is present in M2, M3, Residues 1031237-Sequence at M6, M28 strains and phage 1032011 http://www.sanger.ac.uk/PhiNIH1.1at the NCBI 1e−111 Projects/S_pyogenes 29/11/07/ Bacterium seeHseeL seeM slaB S. equi 0 0 0 0 (beta-haemolytic) S. zooepidemicus NONENONE NONE Contig (beta-haemolytic) zoo122a01.p1k Sequence at Residues175312- http://www.sanger.ac.uk/ 175881 cgi-bin/blast/submitblast/s_zooepidemicus S. pyogenes 1.8e−148 NONE in Manfredo NONE in ManfredoNONE in Manfredo (beta-haemolytic) Residues 204328- Present in variousPresent in various This is present in M2, M3, Sequence at 204943pyogenes strains at pyogenes strains at M6, M28 strains and phagehttp://www.sanger.ac.uk/ NCBI 1e−131 NCBI 1e−125 PhiNIH1.1at the NCBI4e−72 Projects/S_pyogenes 29/11/07/

TABLE 8 other preferred strains Bacterium Disease in animals Disease inhumans S. canis Streptococcal toxic shock urinary infection soft(beta-haemolytic) syndrome in dogs.. Res- tissue infection, piratoryinfection, toxic bacteremia, pneumonia, shock, sepsis in cats boneinfection S. dysgalactiae Mastitis in cattle, subsp dysgalactae S.dysgalactiae septicemia in dogs Respiratory, tissue subsp equisimilisRespiratory disease in infections, cellulitis, (beta-haemolytic) horsessepticemia. S. porcinus Lymphadenitis in pigs, Abortion(beta-haemolytic) abscessation primarily of the head and neck lymphnodes, Abortion

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Sequence Annexes 1-6

Sequence Annex 1—sagA deletion:

WTATGTTACAATTTGCTTCAAATATTTTAGCTACTAGTGTAGCAGAAACAACTCAAGTTGCTCCTGGTGGTTGCTGCTGTTGSHMAPRATGTTACAATTTGCT-----------------------------------------------------------------WTCTGTTCTTGTTGTTGCTGCGTCTCAGCTTCATGGGGCAATACTACCATAAACAACAATTATGGTGCAGCTGAGCCAAAAGSHMAPR-------------------------------------------------------------AAGCTTGCTGAGCCAAAAGWT CGTAA SHMAPR CGTAA The HindIII restriction site used to generate thedeletion construct is shown in italics.Sequence Annex 2—hasA deletion:

WTATGAGAACATTAAAAAACCTCATAACTGTTGTGGCCTTTAGTATTTTTTGGGTACTGTTGATTTACGTCAATGTTTATCTSHMAPRATGAGAACATTAAAAAACCTCATAACTGTTGTGGCCTTTAGTATTTTTTGGGTACTGTTGATTTACGTCAATGTTTATCTWTCTTTGGTGCTAAAGGAAGCTTGTCAATTTATGGCTTTTTGCTGATAGCTTATCTATTAGTCAAAATGTCCTTATCTTTTTSHMAPRCTTTGGTGCTAAAGGAAGCTTGTCAATTTATGGCTTTTTGCTGATAGCTTATCTATTAGTCAAAATGTCCTTATCTTTTTWTTTTACAAGCCATTTAAGGGAAGGGCTGGGCAATATAAGGTTGCAGCCATTATTCCCTCTTATAACGAAGACGCTGAGTCASHMAPRTTTACAAGCCATTTAAGGGAAGGGCTGGGCAATATAAGGTTGCAGCCATTATTCCCTCTTATAACGAAGACGCTGAGTCAWTTTGCTAGAGACCTTAAAAAGTGTTCAGCAGCAAACCTATCCCCTAGCAGAAATTTATGTTGTTGACGATGGAAGTGCTGASHMAPRTTGCTAGAGACCTTAAAAAGTGTTCAGCAGCAAACCTATCCCCTAGCAGAAAT---------------------------WTTGAGACAGGTATTAAGCGCATTGAAGACTATGTGCGTGACACTGGTGACCTATCAAGCAATGTCATTGTTCATCGGTCAGSHMAPR--------------------------------------------------------------------------------WTAGAAAAATCAAGGAAAGCGTCATGCACAGGCCTGGGCCTTTGAAAGATCAGACGCTGATGTCTTTTTGACCGTTGACTCASHMAPR--------------------------------------------------------------------------------WTGATACTTATATCTACCCTGATGCTTTAGAGGAGCTGTTAAAGACCTTTAATGACCCAACTGTTTTTGCTGCGACGGGTCASHMAPR--------------------------------------------------------------------------------WTCCTTAATGTCAGAAATAGACAAACCAATCTCTTAACACGCTTGACAGATATTCGCTATGATAATGCTTTTGGCGTTGAACSHMAPR--------------------------------------------------------------------------------WTGAGCTGCCCAATCAGTTACGGGTAATATCCTTGTTTGCTCAGGCCCACTTAGCGTTTACAGACGCGAGGTGGTTGTTCCTSHMAPR--------------------------------------------------------------------------------WTAATATAGACAGATACATCAACCAGACCTTCCTGGGTATTCCTGTAAGTATCGGTGATGACAGGTGCTTGACCAACTATGCSHMAPR--------------------------------------------------------------------------------WTAACTGATTTAGGAAAGACTGTTTATCAATCCACTGCTAAATGTATTACAGATGTTCCTGACAAGATGTCTACTTACTTGASHMAPR--------------------------------------------------------------------------------WTAGCAGCAAAACCGCTGGAACAAGTCCTTCTTTAGAGAGTCCATTATTTCTGTTAAGAAAATCATGAACAATCCTTTTGTASHMAPR--------GATATCTGGAACAAGTCCTTCTTTAGAGAGTCCATTATTTCTGTTAAGAAAATCATGAACAATCCTTTTGTAWTGCCCTATGGACCATACTTGAGGTGTCTATGTTTATGATGCTTGTTTATTCTGTGGTGGATTTCTTTGTAGGCAATGTCAGSHMAPRGCCCTATGGACCATACTTGAGGTGTCTATGTTTATGATGCTTGTTTATTCTGTGGTGGATTTCTTTGTAGGCAATGTCAGWTAGAATTTGATTGGCTCAGGGTTTTAGCCTTTCTGGTGATTATCTTCATTGTTGCTCTTTGTCGGAACATTCATTACATGCSHMAPRAGAATTTGATTGGCTCAGGGTTTTAGCCTTTCTGGTGATTATCTTCATTGTTGCTCTTTGTCGGAACATTCATTACATGCWTTTAAGCACCCGCTGTCCTTCTTGTTATCTCCGTTTTATGGGGTGCTGCATTTGTTTGTCCTACAGCCCTTGAAATTGTATSHMAPRTTAAGCACCCGCTGTCCTTCTTGTTATCTCCGTTTTATGGGGTGCTGCATTTGTTTGTCCTACAGCCCTTGAAATTGTATWT TCTCTTTTTACTATTAGAAATGCTGACTGGGGAACACGTAAAAAATTATTATAA SHMAPRTCTCTTTTTACTATTAGAAATGCTGACTGGGGAACACGTAAAAAATTATTATAA The EcoRVrestriction site used to generate the deletion construct is shown initalics.Sequence Annex 3—seM deletion:

WTATGTTTTTGAGAAATAACAAGCAAAAATTTAGCATCAGAAAACTAAGTGCCGGTGCAGCATCAGTATTAGTTGCAACAAGSHMAPRATGTTTTTGAGAAATAACAAGCAAAAATTTAGCATCAGAAAACTAAGTGCCGGTGCAGCATCAGTATTAGTTGCAACAAGWTTGTGTTGGGAGGGACAACTGTAAAAGCGAACTCTGAGGTTAGTCGTACGGCGACTCCAAGATTATCGCGTGATTTAAAAASHMAPRTGTGTTGGGAGGGACAACTGTAAAAGCGAACTCTGAGGTTAGTCGTACGGCGACTCCAAGATTATCGCGTGATTTAAAAAWTATAGATTAAGCGAAATAGCCATAAGTAGAGATGCCTCATCAGCCCAAAAAGTTCGAAATCTTCTAAAAGGCGCCTCTGTTSHMAPRATAGATTAAGCGAAATAGCCATAAGTAGAGATGCCTCATCAGCCCAAAAAGTTCGAAATCTTCTAAAAGGCGCCTCTGTTWTGGGGATTTACAGGCATTATTGAGAGGTCTTGATTCAGCAAGGGCTGCGTATGGTAGAGATGATTATTACAATTTATTGGTSHMAPRGGGGATTTACAGGCATTATTGAGAGGTCTTGATTCAGCAAGGGCTGCGTATGGTAGAGATGATTATTACAATTTATTGGTWTGCACCTTTCATCGATGTTAAATGATAAACCTGATGGGGATAGAAGACAATTAAGTTTGGCTTCATTACTTGTAGATGAAASHMAPRGCACCTTCCATCGATGTAAAATGATATC----------------------------------------------------WTTTGAAAAGCGGATTGCTGATGGAGATAGTTATGCAAAACTTCTTGAGGCTAAACTTGCAGCTATTAAATCTCAACAAGAASHMAPR--------------------------------------------------------------------------------WTATGCTTAGAGAAAGAGATTCCCAACTTCGAAATCTAGAGAAGGAAAAAGAACAAGAACTACAAAAAGCTAAAGATGAGCGSHMAPR--------------------------------------------------------------------------------WTTCAAGCTCTTACCGAATCATTCAACAAAACTTTATCAAGATCAACAAAAGAGTATAATAAACTAAAAACAGAACTTGCAASHMAPR--------------------------------------------------------------------------------WTAAGAAAAAGAAAAAGCAGCTAAGATGACTAAGGAATTAGCAGATAAGCTAAGCAATGCTGAAGCAAGTCGTGATAAAGCCSHMAPR--------------------------------------------------------------------------------WTTTTGCAGTATCAAAAGATTTAGCAGATAAACTAAGTAGTGCTGAAGCAAGTCGTGATAAAGCTTTTGCAGTATCAAAAGASHMAPR--------------------------------------------------------------------------------WTTTTAGCAGATAAATTGGCAGCTAAAACAGCAGAAGCTGAAAAGTTAATGGAAAACGTTGGTAGTCTAGACCGCTTGGTAGSHMAPR--------------------------------------------------------------------------------WTAGTCTGCAAAACGTGAAATGGCTCAAAAATTAGCAGAAATTGATCAATTAACTGCTGATAAGGCTAAGGCTGATGCAGAGSHMAPR--------------------------------------------------------------------------------WTCTTGCAGCTGCAAATGACACCATTGCATCACTTCAAACAGAGCTAGAAAAAGCTAAGACAGAGCTTGCTGTTTCAGAGCGSHMAPR--------------------------------------------------------------------------------WTTTTGATTGAATCAGGCAAACGTGAAATTGCTGAGCTACAAAAACAAAAAGATGCTTCTGATAAGGCTTTAGTAGAATCACSHMAPR--------------------------------------------------------------------------------WTAAGCTAATGTAGCAGAGCTTGAAAAACAAAAAGCAGCATCAGATGCTAAGGTAGCAGAGCTTGAAAAAGAAGTTGAAGCTSHMAPR--------------------------------------TGAGATGCTAAGGTAGCAGAGCTTGAAAAAGAAGTTGAAGCTWTGCTAAAGCTGAGGTTGCAGATCTTAAAGTACAATTAGCTAAGAAAGAAGAAGAGCTTGAAGCCGTTAAGAAGGAAAAAGASHMAPRGCTAAAGCTGAGGTTGCAGATCTTAAAGTACAATTAGCTAAGAAAGAAGAAGAGCTTGAAGCCGTTAAGAAGGAAAAAGAWTAGCGCTTGAAGCTAAGATTGAAGAGCTCAAAAAAGCTCATGCTGAGGAACTTTCAAAACTTAAAGAAATGCTTGAGAAGASHMAPRAGCGCTTGAAGCTAAGATTGAAGAGCTCAAAAAAGCTCATGCTGAGGAACTTTCAAAACTTAAAGAAATGCTTGAGAAGAWTAAGACCATGCAAATGCAGATCTTCAAGCAGAAATCAATCGCTTGAAGCAAGAGCTAGCTGACAGGATTAAGTCATTGTCASHMAPRAAGACCATGCAAATGCAGATCTTCAAGCAGAAATCAATCGCTTGAAGCAAGAGCTAGCTGACAGGATTAAGTCATTGTCAWTCAAGGTGGTCGTGCTTCACAAACAAACCCAGGCACTACAACTGCTAAAGCAGGTCAATTGCCATCTACTGGTGAGTCTGCSHMAPRCAAGGTGGTCGTGCTTCACAAACAAACCCAGGCACTACAACTGCTAAAGCAGGTCAATTGCCATCTACTGGTGAGTCTGCWTTAACCCATTCTTCACTATTGCAGCTCTTACTGTCATCGCTGGTGCTGGTATGGCTGTGGTGTCTCCTAAACGCAAAGAAASHMAPRTAACCCATTCTTCACTATTGCAGCTCTTACTGTCATCGCTGGTGCTGGTATGGCTGTGGTGTCTCCTAAACGCAAAGAAAWT ACTAA SHMAPR ACTAA The seM deletion generated two stop codons(underlined), which would abolish cell surface binding of the truncatedSeM product. The EcoRV restriction site used to generate the deletionconstruct is shown in italics.Sequence Annex 4—aroA deletion:

WTATGACACAAACACTTCAGGTTAAGTCTCGTATCAATGACTATCCGATTATCTTTACAGACGATATTTTTCAGCCGCTGAASHMAPRATGACACAAACACTTCAGGTTAAGTCTCGTATCAATGACTATCC------------------------------------WTTCAATTTCTTGCTGAAAAAGGAGACGTCAAGCTATTATTTATCACTGATCAAACGGTATTTGATTTATACCAGCCTTTATSHMAPR--------------------------------------------------------------------------------WTTTAGACGTTTTCAACAGGATTACGATAGTTACCTTCATATTGCTGCTCCAGGGGGGCAATCTAAGTCTCTAGAGGAGGTTSHMAPR--------------------------------------------------------------------------------WTAGTCGGATTTACGATCGACTGATTAGGGCTAATTTTTCTAAAAAGGACGTCATTGTTACTGTTGGAGGAGGGGTGATTGGSHMAPR WTAGATCTTGGGGGATTTGTTGCGGCAACCTTTTACCGCGGGATTTCCTACGTTCAGATTCCAACAACCTTACTTAGTCAGGSHMAPR--------------------------------------------------------------------------------WTTAGACAGCAGCATTGGTGGTAAGGTTGGGGTTCACTTTAAGGGCTTGACCAATATGATAGGCAGTATCTACCCTCCAAACSHMAPR--------------------------------------------------------------------------------WTCAGATTATCGTGTCAGCCAAGTTTTTAGACACGCTTTCTGAAAGAGAATTTGCCTGCGGCATCAGCGAAATGATTAAAATSHMAPR--------------------------------------------------------------------------------WTTGGTTTTATTCATGATCGCAAGCTCTTTCAACAGCTCCTAGCCTTCCCCAAGGACCGCAATCAAGAGCAGCTCAGGCAAASHMAPR--------------------------------------------------------------------------------WTTGATTTTTCAAGCGATTTGCCATAAAAAAAGAGTGGTTGAAAAGGATGAATTTGAAGGCAATCTCCGCATGTCCTTAAATSHMAPR--------------------------------------------------------------------------------WTTTCGGGCATACGCTAGGGCATGCGATTGAAGCCTTATGCCATCACGAGCTTTACAGGCATGGTGAGGCTATTGCGATTGGSHMAPR--------------------------------------------------------------------------------WTCATGGTCTTTGAGGCCAAGCTGGCCGTCCAGCAGCAGCTATTGAGCCAACAGGATTTAGAGGCATTACAGGCTGCCTTTGSHMAPR--------------------------------------------------------------------------------WTAGGCTTATCAGCTACCTACCACACTTGAGGCTAAGTCAATGACAGCCGAAGCCTTGATGACTGTTTTAAAAACAGATAAGSHMAPR--------------------------------------------------------------------------------WTAAAAATTCTGGTCAGCATATTGTCCTCATTTTGCCAACGACAAAAGGCTATGTAAGCTTTCCTATTGCTAAGCATGACAGSHMAPR--------------------------------------------------------------------------------WT TCGCCTGCTGGATTGGCTAAGAAGCCTGCTAGATATCGCCTGA SHMAPR-------------------------------GATATCGCCTGA The EcoRV restriction siteused to generate the deletion construct is shown in italics.Sequence Annex 5—pyrC deletion:

WTATGATATCAGGGATCAAGACAGTTACGTCCGATATGTCAAGCAAAACAAATAATCACTGCCTAGATAAATCAGAAATTGCSHMAPRATGATATCAGGGATCAAGACAGTTACGTCCGATATGTCAAGCAAAACAAATAATCACTGCCTAGATAAATCAGAAATTGCWTTAGGGTTATGCTTGATTATCCTGATAAGCAGATAAGTAGATTTGACATAGGAGGGGTCATGTTATTAATTAAAAATGGGCSHMAPRTAGGGTTATGCTTGATTATCCTGATAAGCAGATAAGTAGATTTGACATAGGAGGGGTCATGTTATTAATTAAAAATGGGCWTGTGTGATGGATCCAAAATCACAGCTAGATCAGGTGGCAGATGTCTTAATTGATAATGGAAGGATTTTACGGATTGCTCCASHMAPRGTGTGATGGATCCAAAATCACAGCTAGATCAGGTGGCAAGCTT-------------------------------------WTGACATTGAGCATGATGAGGTAGAGCAGATCGATGCCAGTGGACTTGTTGTTGCTCCTGGTTTAGTGGATATTCATGTTCASHMAPR--------------------------------------------------------------------------------WTTTTTAGAGAGCCGGGTCAAACGCACAAGGAGGACATTCATACAGGTGCTCTGGCAGCAGCTGCTGGTGGGGTGACAACAGSHMAPR--------------------------------------------------------------------------------WTTAGTCATGATGGCAAACACCAATCCTGTTATATCAGATACGGAAACCTTACAGGCTGTTCTAGCAAGTGCTGCTAAAGAASHMAPR--------------------------------------------------------------------------------WTAAAATTAACATTTATACCAATGCTAGTGTGACCAAGCGGTTCAATGGCCAAGAGCTAACAGACTTTAAAGCGCTCTTAGCSHMAPR--------------------------------------------------------------------------------WTAGCTGGTGCGGTCAGTTTTTCTGATGATGGCATTCCTTTAGAGAGCTCCAAGGTCTTAAAGGAAGCATTGGATTTGGCTASHMAPR--------------------------------------------------------------------------------WTAGGCCAACAAGACCTTCATTGCCCTGCATGAGGAGGATCCCCAATTAAACGGTGTCCTTGGCTTCAATGAGCATATCGCTSHMAPR--------------------------------------------------------------------------------WTAAGGATCATTTTCATTTTTGTGGCGCTACTGGTGTGGCAGAATATAGTATGATTGCCAGAGATGTGATGATTGCCTATGASHMAPR--------------------------------------------------------------------------------WTTCGACAAGCTCATGTTCATATTCAACATTTATCTAAGGCTGAGTCTGTTAAGGTAGTTGCCTTTGCTCAGCAGTTAGGTGSHMAPR--------------------------------------------------------------------------------WTCCAAGGTCACAGCCGAGGCAACACCGCAGCATTTTTCTAAAACAGAAGACCTTTTACGGCTTGCAGGGGCAAATGCCAAGSHMAPR--------------------------------------------------------------------------------WTATGAATCCGCCTCTAAGAACAGAACAAGATAGATTAGCAGTTATTGAGGGGCTCAAATCAGGTGTCATAGCTATTATTGCSHMAPR--------------------------------------------------------------------------------WTAACGGATCATGCACCACATCATCGTGATGAAAAGGCCGTTGCTGATCTGACCAAGGCACCATCTGGAATGACCGGCTTAGSHMAPR--------------------------------------------------------------------------------WTAAACCTCATTGTCATTAGGCCTGACAAATCTTGTGGAGCCGAGCCATCTTTCATTGATGGCGTTATTAGAGAAAATGACCSHMAPR--------------------------------------------------------------------------------WTATTAATCCAGCCTCACTATATAGCTTTGATGCTGGTTATTTGGCTGAGTCTGGCCCTGCTGATCTTGTTATTTTTGCTGASHMAPR--------------------------------------------------------------------------------WTCAAGGAGGAGCGTTTGGTAACAGAAGCCTTTGCTTCAAAGGCTAGTAATTCACCTTTTATTGGCGAAACCCTAAAGGGAGSHMAPR--------AGCGTTTGGTAACAGAAGCCTTTGCTTCAAAGGCTAGTAATTCACCTTTTATTGGCGAAACCCTAAAGGGAGWT TTGTGAAATACACCATTGCTAAGGGACAAATTGTTTATCAGGCAGACAACTAA SHMAPRTTGTGAAATACACCATTGCTAAGGGACAAATTGTTTATCAGGCAGACAACTAA The HindIIIrestriction site used to generate the deletion construct is shown initalics.Sequence Annex 6—recA deletion:

WTTTGGCAAAAAAAGTTAAAAAAAATGAAGAAATCACCAAAAAATTTGGTGATGAACGTCGTAAAGCACTTGATGATGCGTTSHMAPRTTGGCAAAAAAAGTTAAAAAAAATGAAGAAATCACCAAAAAATTTGGTGATGAACGTCGTAAAGCACTTGATGATGCGTTWTAAAGAACATCGAAAAAGATTTTGGTAAGGGTGCGGTTATGCGCCTTGGTGAGCGTGCAGAGCAAAAGGTTCAGGTGATGASHMAPRAAAGAACATCGAAAAAGATTTTGGTAAGGGTGCGGTTATGCGCCTTGGTGAGCGTGCAGAGCAAAAGGTTCAGGTGATGAWTGTTCAGGCAGTCTTGCTTTAGACATTGCGCTTGGAGCAGGTGGCTATCCTAAAGGGCGTATTATTGAAATCTATGGACCASHMAPRGTTCAGGCAGTCTTGCTTTAGACATTGCGCTTGGAGCAGGTGGCTATCCTAAAGGGCGTATTATTGAAATCTATGGACCAWTGAGTCTTCTGGTAAAACAACAGTTGCCCTGCATGCAGTAGCGCAGGCTCAAAAAGAAGGTGGTATTGCAGCCTTCATTGASHMAPRGAGTCTTCTGGTAAAACAACAGTTGCCCTGCATGCAGTAGCGCAGGCTCAAAAAGAAGGTGGTATTGCAGCCTT------WTTGCGGAGCATGCCTTGGACCCTGCTTATGCTGCGGCGCTGGGTGTTAATATTGATGAGCTGCTTTTGTCACAGCCGGATTSHMAPR--------------------------------------------------------------------------------WTCTGGTGAGCAAGGACTTGAGATAGCAGGTAAACTGATTGATTCTGGTGCTGTTGATTTGGTTGTTGTCGACTCTGTTGCASHMAPR--------------------------------------------------------------------------------WTGCTCTAGTGCCTCGTGCTGAGATTGATGGTGATATTGGTGATAACCATGTTGGCTTGCAGGCTCGTATGATGAGTCAGGCSHMAPR--------------------------------------------------------------------------------WTGATGCGTAAGCTTTCAGCCTCAATCAATAAAACCAAGACAATTGCGATCTTTATTAACCAGCTGCGTGAAAAGGTAGGGGSHMAPR--------------------------------------------------------------------------------WTTTATGTTTGGTAATCCAGAGACGACACCAGGTGGTCGTGCTTTGAAATTCTATGCCTCTGTCCGTCTGGATGTTCGTGGASHMAPR--------------------------------------------------------------------------------WTACAACACAAATAAAAGGAACTGGAGATCAAAAAGACAGTAGTATTGGTAAGGAAACCAAGATTAAGGTTGTTAAGAATAASHMAPR--------------------------------------------------------------------------------WTGGTTGCTCCGCCATTTAAGGTGGCTGAGGTTGAAATCATGTATGGAGAAGGCATCTCACGTACAGGTGAGCTGATTAAAASHMAPR----------------------------------------GATATCGAAGGCATCTCACGTACAGGTGAGCTGATTAAAAWTTTGCTTCAGATTTAGACATTATTCAAAAGGCTGGTGCTTGGTTCTCTTATAACGGTGAAAAAATTGGTCAGGGCTCTGAASHMAPRTTGCTTCAGATTTAGACATTATTCAAAAGGCTGGTGCTTGGTTCTCTTATAACGGTGAAAAAATTGGTCAGGGCTCTGAAWTAATGCCAAGAGATATTTGGCTGATCACCCAGAGCTGTTTGATGAGATTGACCATAAGGTGCGTGTTAAATTTGGCTTGCTSHMAPRAATGCCAAGAGATATTTGGCTGATCACCCAGAGCTGTTTGATGAGATTGACCATAAGGTGCGTGTTAAATTTGGCTTGCTWTTGAAGATACTGAGGAAAGTGCAGCTGCAGATACAGTTGCAGCCAAAGCAGATGAGTTGGTTTTAGAGCTAGACGATGCCASHMAPRTGAAGATACTGAGGAAAGTGCAGCTGCAGATACAGTTGCAGCCAAAGCAGATGAGTTGGTTTTAGAGCTAGACGATGCCAWT TTGAAATTGAGGATTAG SHMAPR TTGAAATTGAGGATTAG The EcoRV restriction siteused to generate the deletion construct is shown in italics.

1-43. (canceled)
 44. A Streptococcus vaccine strain comprising aStreptococcus strain with the following modifications in its genome: (i)attenuation of one or more essential biosynthetic genes, (ii)attenuation of one or more genes that encode a hemolytic toxin, orprotein involved in the production thereof, plus any one, two, or threeof the following modifications: (iii) attenuation of one or more genesthat encode a protein responsible for immune evasion (iv) modificationof one or more genes to permit serological discrimination of the vaccinestrain based on analysis of a protein encoded by the genes, and (v)attenuation of one or more genes that encode an enzyme responsible forrecombination repair of the genome.
 45. The strain of claim 44 whereinthe attenuation is deletion.
 46. The strain of claim 44 wherein twoessential biosynthetic genes are attenuated and wherein the biosyntheticgenes are selected from the group consisting of genes in the aromatic orpyramidine or purine pathways.
 47. The strain of claim 46 wherein thebiosynthetic genes are aroB and pyrC.
 48. The strain of claim 44 whereinthe gene encoding a hemolytic toxin, or protein involved in theproduction thereof, is the sagA gene.
 49. The strain of claim 44 whereinthe protein responsible for immune evasion is one that is responsiblefor formation of a hyaluronate capsule.
 50. The strain of claim 49wherein the protein is encoded by the hasA gene.
 51. The strain of claim44 wherein the protein responsible for immune invasion is a superantigenencoded by a gene selected from the group consisting of seeH, seeI, seeLand seeM.
 52. The strain of claim 44 wherein the protein responsible forimmune invasion is a phospholipase encoded by slaA or slaB.
 53. Thestrain of claim 44 wherein the gene encoding the M protein in the strainor wherein one or more of the superantigen genes, selected from thegroup consisting of seeH, seeI, seeL and seeM, is modified to permitserological discrimination.
 54. The strain of claim 44 wherein theenzyme responsible for recombination repair of the genome is encoded bythe recA gene.
 55. The strain of claim 44 wherein the Streptococcus is aBeta-hemolytic streptococcus shown in Table 7a, Table 7b or Table
 8. 56.The strain of claim 55 wherein the Streptococcus is S. equi.
 57. Thestrain of claim 44 wherein modifications (i) and (ii) are combined with(iii), (iv) and (v).
 58. The strain of claim 57, wherein the strain is aSHMAPR strain and wherein the modifications are attenuations in the sagAgene; the hasA gene; the gene encoding the M-protein; the aroB and pyrCgenes; and the recA gene
 59. The strain of claim 58, wherein the strainis a SHMAPR strain comprising further attenuations one, two, three,four, five or six of the following genes: seeH, seeI, seeL, seeM, slaA,and slaB.
 60. The S. equi strain SHMAPR deposited under accession number13412 with the NCTC.
 61. The deposited strain of claim 60 in amicrobiological pure bacterial culture.
 62. A process for producing aStreptococcus vaccine strain comprising (a) making the followingmodifications to the genome of the strain: (i) attenuation of one ormore essential biosynthetic genes, (ii) attenuation of one or more genesencoding a hemolytic toxin or protein involved in the productionthereof, plus any one, two or three of the following modifications:(iii) attenuation of one or more genes that encode a protein responsiblefor immune evasion, (iv) modification of one or more genes to permitserological discrimination of the vaccine strain based on analysis of aprotein encoded by the genes, and (v) attenuation of one or more genesthat encode an enzyme responsible for recombination repair of thegenome, and (b) culturing the resulting strain.
 63. The process of claim62, which does not incorporate heterologous plasmid DNA or antibioticresistance genes into the resulting vaccine strain.
 64. The process ofclaim 62 wherein the genes are selected from the following genes: (i)the essential biosynthetic genes aroB and pyrC; (ii) the sagA geneencoding the hemolytic toxin or protein involved in the productionthereof; (iii) a gene that encodes the protein responsible for immuneevasion, selected from the group consisting of hasA, seeH, seeI, seeL,seeM, slaA, and slaB; (iv) a gene that permits serologicaldiscrimination of the vaccine strain based on analysis of a proteinencoded by the gene, which is selected from the group consisting of thegene encoding the M protein, seeH, seeI, seeL and seeM; and (v) the recAgene, which encodes the enzyme responsible for recombination repair ofthe genome.
 65. The process of claim 64 wherein the Streptococcus strainis shown in Table 7a, Table 7b or Table
 8. 66. A process for preparing avaccine, comprising combining the strain of claim 44 with apharmaceutically acceptable carrier and one or more further additivesselected from a) one or more stabilizing proteins to facilitatefreeze-drying of the vaccine; b) an adjuvant; and c) a differentattenuated pathogen or antigenic material from a different pathogen inorder to provide a multivalent vaccine.
 67. The vaccine strain of claim44, further comprising a pharmaceutically acceptable carrier.
 68. Amethod for immunizing a subject against a Streptococcal pathogen,comprising administering to the subject the Streptococcus vaccine strainof claim 67 in an amount sufficient to produce a protective immuneresponse thereto.
 69. The method of claim 68 wherein the streptococcusis S. equi, and the vaccine strain inhibits Streptococcus equi orStreptococcus zooepidemicus infection in horses.
 70. The method of claim69 wherein the infection is strangles.
 71. The method of claim 70wherein the strain is administered intramuscularly or intranasally. 72.The method of claim 71 where the strain is a live strain.